HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Ikawa, T. Articles by Oida, S. Search for Related Content PubMed PubMed Citation Articles by Ikawa, T. Articles by Oida, S.

Porcine Amelogenin is Expressed from the X and Y Chromosomes

¹ Department of Biochemistry and
² Department of Periodontology, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama, 230-8501, Japan; and
³ Dental Research Laboratory, University of Michigan, 1210 Eisenhower Place, Ann Arbor, MI 48108, USA;

^* corresponding author, oida-s{at}tsurumi-u.ac.jp

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Amelogenin is the major enamel matrix component in developing teeth. In eutherian mammals, amelogenin is expressed from the X chromosome only, or from both the X and Y chromosomes. Two classes of porcine amelogenin cDNA clones have been characterized, but the chromosomal localization of the gene(s) encoding them is unknown. To determine if there are sex-based differences in the expression of porcine amelogenin, we paired PCR primers for exons 1a, 1b, 7a, and 7b, and amplified enamel organ-derived cDNA separately from porcine males and females. The results show that exons 1a/2a and 7a are always together and can be amplified from both males (XY) and females (XX). Exons 1b/2b and 7b are also always paired, but can be amplified only from females. We conclude that porcine amelogenin is expressed from separate genes on the X and Y chromosomes, and not, as previously proposed, from a single gene with two promoters.

KEY WORDS: enamel • amelogenin • amelogenesis imperfecta • AMELX • sex chromosomes

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Amelogenins are tissue-specific proteins that are primarily expressed by cells derived from the inner enamel epithelium of tooth organs, and represent the major enamel matrix constituent in developing teeth. In humans, there are 2 amelogenin genes, located on the sex chromosomes (AMELX and AMELY). Approximately 90% of amelogenin mRNA expression is from the X chromosome (Salido et al., 1992). Mutations in AMELX cause X-linked amelogenesis imperfecta (Lagerström et al., 1991), indicating that AMELX is critical for proper dental enamel formation. The X and Y chromosomal copies of the human amelogenin genes are non-allelic, and do not undergo homologous recombination. This feature has proved advantageous for sex determination in forensic science, where oligonucleotide primer pairs are used that give different sizes of PCR amplification products for the X and Y amelogenin genes (Akane et al., 1992). There is a small failure rate for the amelogenin sex test, however, due to the rare deletion of AMELY (Steinlechner et al., 2002), which can reach as high as 0.9 to 3.6% in certain ethnic groups in Malaysia and India (Chang et al., 2003). While there have been no studies on whether the enamel formed in the absence of AMELY is defective, the low expression of AMELY relative to AMELX and the absence of AMELY in assorted mammalian species, including rodents (Lau et al., 1989) and selected primates (Nakahori et al., 1991), suggest that AMELY is not critical for proper dental enamel formation.

Amelogenin sequences have been cloned from the developing teeth of several non-mammalian vertebrate species (Toyosawa et al., 1998), but most of what is known comes from mammals. Mammals are divided into 3 groups, based upon their means of reproduction (Macdonald, 1984). The prototheria are egg-laying (monotremes), the metatheria are non-placental (marsupials), and the eutheria are placental mammals. The genomic positions of amelogenin genes in amphibians and reptiles are unknown. In monotremes and marsupials, there is evidence that the amelogenin gene might be autosomal (Watson et al., 1992). In eutherian mammals, the amelogenin gene is on the X chromosome, or on both the X and Y chromosomes, depending upon the species (Simmer and Snead, 1995). Previously, 2 types of amelogenin clones (designated a and b) were isolated from a porcine cDNA library (Hu et al., 1996). These clones showed no nucleotide differences in their coding regions for the secreted protein. This pattern is very different from that of the amelogenin coding regions for bovine and human amelogenin, where there are significant differences in the proteins expressed from the X and Y chromosomes. The 100% nucleotide identity in exons 3 through 6 of the 2 types of pig amelogenin cDNAs suggested that, in the pig, there might be a single amelogenin gene that is expressed from 2 promoters (Hu et al., 1996). In this study, we determined whether there are sex-based differences in the expression of porcine amelogenin, and here we discuss the results in the light of recent discoveries concerning the evolution of the amelogenin gene (Iwase et al., 2001, 2003).

	MATERIALS & METHODS

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
All animal materials used in this study were obtained by methods in accordance with and approved by the institutional review board at Tsurumi University in Japan.

RT-PCR of RNA Isolated from Pig Enamel Organ Epithelia
Mandibular incisors were extracted from six-month-old male and female pigs obtained from a slaughterhouse. RNA was extracted from the enamel organ epithelia (EOE) by means of the RNA isolation kit and protocol (Biotecx Laboratories, Houston, TX, USA). The reverse-transcription (RT) reaction used the You-prime First-strand Beads (Amersham Biosciences, Piscataway, NJ, USA). The amelogenin PCR primers included 3 sense primers-(P1a) 5'-ACAAA CTTACTCTGAATACGTA-3', (P1b) 5'-GATCAGACCATGAGAAGAG AAC-3', and (P5) 5'-ACCCCTCYG AAGTGGTACCAG-3'and 3 anti-sense primers-(P2) 5'-CAAGAAT GGGACCTGGATT-3', (P7a) 5'-GATCAGACCATGAGAAGA GAAC-3', and (P7b) 5'-GAGAAAA CTAAATTAGACATTTC-3'. Primers P1a and P1b are specific for exons 1a and 1b, respectively. Primer P2 is a common anti-sense primer for exon 2. Primer P5 is a common sense primer for exon 5, and primers P7a and P7b are specific for exons 7a and 7b, respectively (Hu et al., 1996). A 20-µL vol of RT product was amplified with the Taq DNA polymerase and the 8 possible combinations of sense and anti-sense primers. The reactions had a 10-minute denaturation at 94°C, followed by 32–35 cycles each, with denaturation at 94°C for 30 sec, primer annealing at 55°C for 30 sec, and product extension at 72°C for 30 sec. In the final cycle, the 72°C extension was for 7 min.

PCR Amplification of Genomic DNA
Genomic DNA was obtained from male and female pig enamel organs. Fresh pig enamel organs were cut into small pieces, and purified with use of the QIA amp Tissue Kit (Qiagen, Valencia, CA, USA). Genomic DNA (0.1 µg) was used as a template for each PCR reaction and was amplified with specific primer sets (P1a/P2 and P1b/P2). PCR amplification was a five-minute denaturation at 94°C, followed by 40 cycles each with denaturation at 94°C for 50 sec, primer annealing at 58°C for 30 sec, and product extension at 68°C for 5 min. The final cycle, the 70°C extension, was for 10 min.

Preparation of Immature Enamel Matrix
The method for preparation of the enamel samples has been described previously (Fukae et al., 1993). Briefly, permanent incisor tooth germs were dissected from the fresh mandibles of male and female pigs. The soft tissue was removed, and the outer, secretory-stage enamel was scraped from the surface.

SDS-Polyacrylamide Gel Electrophoresis
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with a 15% polyacrylamide slab gel (Atto Corporation, Tokyo, Japan) according to the method of Laemmli (1970). The lyophilized protein samples were dissolved in 10 mM Tris-HCl buffer (pH 8.5) containing 1% SDS and 0.192 M glycerol at a concentration of 0.1% w/v. Electrophoresis was carried out with a current of 23 mA for 1–2 hrs. The gels were stained with 0.125% Coomassie Brilliant Blue (CBB) and de-stained with 7.5% acetic acid/5% methanol solution, or, alternatively, were stained with Stains-all. The apparent molecular weights of the protein bands were estimated based on Bio-Rad LMW protein standards.

Phylogenetic Analysis
Amelogenin coding sequences were aligned with the use of ClustalW (Thompson et al., 1994). The best tree was generated by the neighbor-joining (NJ) method with the Kimura two-parameter correction for differences in the rates of transition and transversion mutations and for multiple substitutions at each site, and taking into account positions with gaps (Kimura, 1980; Saitou and Nei, 1987; Perrière and Gouy, 1996). The tree was constructed with the use of TreeView 1.6.6 (Page, 1996). The out groups were defined as Xenopus laevis 1 and 2.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Porcine cDNA and genomic DNA were prepared from secretory-stage EOE dissected from male and female developing incisors, and used as template for PCR amplification. Sense primers specific for exons 1a and 1b were paired with anti-sense primers specific for exons 7a and 7b and used for RT-PCR. We used the 4 possible combinations of these primers separately to amplify cDNA prepared from porcine males and females. The primer annealing sites are shown on the model structure for the pig amelogenin gene (Fig. 1A). Amelogenin amplification products were observed when primer P1a was paired with P7a, and when primer P1b was paired with P7b, but no amplification products were observed when an "a" primer was paired with a "b" primer (Fig. 1B). The primer pair 1a/7a amplified cDNA from both males and females; however, the primer pair 1b/7b amplified cDNA only from males. Characterization of the PCR amplification products by DNA sequencing demonstrated that the 2 bands observed in each lane corresponded to alternatively spliced amelogenin transcripts encoding P173 (exons 1-2-3-5-6-7) and P56 (exons 1-2-3-5-6s-7), which is known as the leucine-rich amelogenin protein, or LRAP (Fincham et al., 1981). RT-PCR was also performed with an exon 5 sense primer (P5) paired with anti-sense primers (P7a and P7b) specific for exons 7a and 7b, respectively (Fig. 1C). Characterization of the PCR amplification products by DNA sequencing demonstrated that the 2 bands observed in each lane were derived from alternatively spliced amelogenin transcripts encoding P173 (exons 5-6-7) and P56 (exons 5-6s-7).

View larger version (42K): [in this window] [in a new window]   Figure 1. Model structure and PCR products of porcine amelogenin. (A) Model showing the intron-exon structure of the porcine amelogenin genes. The lines correspond to introns and the bars to exons. The white bars indicate the regions with the same sequence; arrows indicate oligonucleotide annealing sites. (B) Polyacrylamide gel (4.5%) stained with ethidium bromide showing PCR products observed when amplifying cDNA template and using amelogenin exon 1- and exon 7-specific primer sets. (C) PCR products observed when amplifying cDNA template and using amelogenin exon 5- and exon 7-specific primer sets. Lane M is the molecular marker (HpaI digest of x174 phage). (D) Agarose gel (1%) stained with ethidium bromide showing PCR products observed when amplifying genomic DNA with amelogenin exon 1- and exon 2-specific primer sets. The amplification products were separated on 1%. Lane M is molecular marker (HindIII digest of phage).

We carried out SDS-PAGE analysis to determine if sex-based differences in amelogenin protein could be distinguished in the developing teeth of males and females (Fig. 2). Enamel proteins were extracted from developing incisors obtained from male and female pigs and characterized by SDS-PAGE analyses. No differences were observed in the electrophoretic patterns of enamel proteins from porcine males and females, whereas differences were evident when similar analyses were performed with human enamel extracts (Fincham et al., 1991).

View larger version (103K): [in this window] [in a new window]   Figure 2. SDS-PAGE patterns of enamel protein extracted from male and female incisors. The left gel is stained with CBB, and the right is stained with Stains-all. Lane M is molecular marker, lane 1 shows enamel proteins from males, and lane 2 shows enamel proteins from females.

View larger version (25K): [in this window] [in a new window]   Figure 3. Phylogenetic tree and revised model structure of amelogenin genes. (A) Phylogenetic tree of cDNA sequences of amelogenin (signal peptide and protein coding region). Numbers at forks indicate percentage of 1000 bootstrap re-samplings that support tree topological elements. (B) Revised model structure of male and female porcine amelogenin genes. The 7 amelogenin exons are indicated by boxes. Unfilled boxes correspond to the amelogenin coding region; patterned boxes correspond to exonic non-coding regions. The DNA sequences in the X and Y copies of the porcine amelogenin genes are identical in the coding regions for the secreted protein (solid line), but differ in the coding regions for the signal peptides (dashed line).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

The hypothesis that pig amelogenin is expressed from a single gene with 2 promoters, rather than 2 separate genes on the X and Y chromosomes (Hu et al., 1996), was based upon the 100% nucleotide identity in exons 3, 5, and 6, which encode 170 of the 173 amino acids of the major pig amelogenin isoform (P173). In contrast, this same region of the 2 bovine amelogenin genes varies at 8 positions in exon 5 and 25 positions in exon 6, not including the 63-basepair deletion in exon 6. If the X and Y copies were present in the common bovine and porcine ancestor, we would expect a similar degree of divergence between the 2 sequences in these exons. In this report, we disprove the one-gene/two-promoter hypothesis and demonstrate that porcine amelogenin is expressed from separate genes on the X and Y chromosomes. Understanding how the porcine X and Y amelogenin genes could show such high coding identity downstream of exon 2, when compared with the X and Y copies of the bovine and human genes, comes from recent insights about the evolution of amelogenin genes.

Amelogenin genes appear to have translocated to the X and Y chromosomes in a species ancestral to all eutherians, perhaps 80 to 130 million years ago (mya). The earliest sex-linked amelogenin genes apparently localized within the pseudo-autosomal region on the short arms of the sex chromosomes. These ancestral X and Y copies of the amelogenin genes would have undergone homologous recombination, which maintained sequence identity between the 2 alleles. After translocation to the sex chromosomes, the non-recombining portion of the short arm of the sex chromosomes expanded so that AMELX and AMELY spanned the pseudo-autosomal boundary. Homologous recombination of the amelogenin gene no longer occurred upstream of a transposon located within intron 2, but recombination downstream of intron 2 continued (Iwase et al., 2001, 2003). After joining the non-recombining regions of the sex chromosomes, the 5' regulatory regions of AMELX and AMELY began to diverge. This appears to be the state of the amelogenin gene at the start of the great mammalian radiation in the early Cenozoic era (~ 65 mya), which caused the large-scale isolation of mammalian amelogenin genes through speciation. Then, independently and at different times in different mammalian lines, AMELX and AMELY as a whole joined the non-recombining region of the sex chromosomes, making them non-allelic, and causing them to drift apart. By definition, the amelogenin genes became part of the non-recombining region because of suppression of homologous recombination between AMELX and AMELY during male meiosis. In the primate line, a possible cause of this suppression was a Y chromosomal inversion that flipped AMELY relative to AMELX, which occurred about 30 to 50 mya (Lahn and Page, 1999). AMELX is still located close to the pseudo-autosomal boundary, which, in mice, was identified by chromosomal ‘walking’ from the amelogenin gene (Palmer et al., 1997). Because the mammalian radiation occurred at a time when AMELX and AMELY were still partly allelic (maintaining sequence identity from exons 3 through 7), the coding regions for AMELX and AMELY from a given species generally show more similarity to each other than they do for amelogenin genes from other mammals (Fig. 3A). Previously, this phylogenetic pattern was interpreted to indicate that AMELX has repeatedly duplicated and translocated to the Y chromosome in different mammalian lines (Simmer and Snead, 1995). It now seems more likely that AMELX and AMELY were both present at the beginning of the mammalian radiation, but started to diverge at different times as AMELX and AMELY independently joined the non-recombination portions of the sex chromosomes.

In general, gene inversions do not appear to be the principle mechanism that ended AMELX and AMELY recombination. The suppression of recombination between AMELX and AMELY appears to have have occurred more gradually than could be explained by the inversion hypothesis (Marais and Galtier, 2003). Sophisticated region-by-region analyses of the growing database of amelogenin genes suggest that the pseudo-autosomal boundary (PAB) crept across the amelogenin gene in a series of steps (Iwase et al., 2001, 2003; Marais and Galtier, 2003). This movement of the PAB was presumably due to alterations in sequences or structural signals responsible for initiating or suppressing recombination (Iwase et al., 2001).

Because of the convoluted history of amelogenin genes losing their ability to undergo homologous recombination, sequence variations between AMELX and AMELY are useful for sex determination, but these tests are different for each species. For instance, a single PCR primer pair that spans a length variation in intron 1 is used for sex determination in humans (Sullivan et al., 1993), while a length variation in intron 5 is used in cattle (Chen et al., 1999). Still another sequence variation is used in the Japanese black bear (Yamamoto et al., 2002), while the adjacent Y-linked SRY locus must be included in the amplification for sex determination in orangutans (Steiper and Ruvolo, 2003).

	ACKNOWLEDGMENTS

 
This work was supported by grant-in-aid No. 12671818, funding from Bio-Venture and the High Technology Research Centers Project from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and by USPHS Research Grant DE-11301 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA. These sources constitute the totality of funding for this project.

Received March 16, 2004; Last revision November 12, 2004; Accepted November 17, 2004

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

 
Akane A, Seki S, Shiono H, Nakamura H, Hasegawa M, Kagawa M, et al. (1992). Sex determination of forensic samples by dual PCR amplification of an X-Y homologous gene. Forensic Sci Int 52:143–148.[ISI][Medline]

Chang YM, Burgoyne LA, Both K (2003). Higher failures of amelogenin sex test in an Indian population group. J Forensic Sci 48:1309–1313.[ISI][Medline]

Chen CM, Hu CL, Wang CH, Hung CM, Wu HK, Choo KB, et al. (1999). Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene. Mol Reprod Dev 54:209–214.[ISI][Medline]

Fincham AG, Belcourt AB, Termine JD, Butler WT, Cothran WC (1981). Dental enamel matrix: sequences of two amelogenin polypeptides. Biosci Rep 1:771–778.[ISI][Medline]

Fincham AG, Bessem CC, Lau EC, Pavlova Z, Shuler C, Slavkin HC, et al. (1991). Human developing enamel proteins exhibit a sex-linked dimorphism. Calcif Tissue Int 48:288–290.[ISI][Medline]

Fukae M, Tanabe T, Uchida T, Yamakoshi Y, Shimizu M (1993). Enamelins in the newly formed bovine enamel. Calcif Tissue Int 53:257–261.[ISI][Medline]

Hu C-C, Bartlett JD, Zhang CH, Qian Q, Ryu OH, Simmer JP (1996). Cloning, cDNA sequence, and alternative splicing of porcine amelogenin mRNAs. J Dent Res 75:1735–1741.[Abstract/Free Full Text]

Iwase M, Satta Y, Takahata N (2001). Sex-chromosomal differentiation and amelogenin genes in mammals. Mol Biol Evol 18:1601–1603.[Free Full Text]

Iwase M, Satta Y, Hirai Y, Hirai H, Imai H, Takahata N (2003). The amelogenin loci span an ancient pseudoautosomal boundary in diverse mammalian species. Proc Natl Acad Sci USA 100:5258–5263.[Abstract/Free Full Text]

Kimura M (1980). A simple method for estimating evolutionary rates of base substitution through comparative studies of nucleotide sequences. J Mol Evol 16:111–120.[ISI][Medline]

Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.[Medline]

Lagerström M, Dahl N, Nakahori Y, Nakagome Y, Bäckman B, Landegren U, et al. (1991). A deletion in the amelogenin gene (AMG) causes X-linked amelogenesis imperfecta (AIH1). Genomics 10:971–975.[ISI][Medline]

Lahn BT, Page DC (1999). Four evolutionary strata on the human X chromosome. Science 286:964–967.[Abstract/Free Full Text]

Lau EC, Mohandas T, Shapiro LJ, Slavkin HC, Snead ML (1989). Human and mouse amelogenin gene loci are on the sex chromosomes. Genomics 4:162–168.[ISI][Medline]

Macdonald D, editor (1984). The encyclopedia of mammals. New York: Facts on File, Inc.

Marais G, Galtier N (2003). Sex chromosomes: how X-Y recombination stops. Curr Biol 13:R641–R643.[ISI][Medline]

Nakahori Y, Takenaka O, Nakagome Y (1991). A human X-Y homologous region encodes amelogenin. Genomics 9:264–269.[ISI][Medline]

Page RDM (1996). TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358.[Free Full Text]

Palmer S, Perry J, Kipling D, Ashworth A (1997). A gene spans the pseudoautosomal boundary in mice. Proc Natl Acad Sci USA 94:12030–12035.[Abstract/Free Full Text]

Perrière G, Gouy M (1996). WWW-Query: an on-line retrieval system for biological sequence banks. Biochimie 78:364–369.[Medline]

Saitou N, Nei M (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425.[Abstract]

Salido EC, Yen PH, Koprivnikar K, Yu L-C, Shapiro LJ (1992). The human enamel protein gene amelogenin is expressed from both the X and Y chromosomes. Am J Hum Gen 50:303–316.[ISI][Medline]

Simmer JP, Snead ML (1995). Molecular biology of the amelogenin gene. In: Dental enamel: formation to destruction. Robinson C, Kirkham J, Shore R, editors. Boca Raton: CRC Press, pp. 59–84.

Steinlechner M, Berger B, Niederstatter H, Parson W (2002). Rare failures in the amelogenin sex test. Int J Legal Med 116:117–120.[ISI][Medline]

Steiper ME, Ruvolo M (2003). Genetic sex identification in orangutans. Anthropol Anz 61:1–5.[Medline]

Sullivan KM, Mannucci A, Kimpton CP, Gill P (1993). A rapid and quantitative DNA sex test: fluorescence-based pcr analysis of X-Y homologous gene amelogenin. BioTechniques 15:636–642.[ISI][Medline]

Thompson J, Higgins D, Gibson T (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680.[Abstract/Free Full Text]

Toyosawa S, O’hUigin C, Figueroa F, Tichy H, Klein J (1998). Identification and characterization of amelogenin genes in monotremes, reptiles and amphibians. Proc Natl Acad Sci USA 95:13056–13061.[Abstract/Free Full Text]

Watson JM, Spencer JA, Graves JAM, Snead ML, Lau EC (1992). Autosomal localization of the amelogenin gene in monotremes and marsupials: implications for mammalian sex chromosome evolution. Genomics 14:785–789.[ISI][Medline]

Yamamoto K, Tsubota T, Komatsu T, Katayama A, Murase T, Kita I, et al. (2002). Sex identification of Japanese black bear, Ursus thibetanus japonicus, by PCR based on amelogenin gene. J Vet Med Sci 64:505–508.[ISI][Medline]

This article has been cited by other articles:

				L. Xu, H. Harada, and A. Taniguchi The Exon 6ABC Region of Amelogenin mRNA Contribute to Increased Levels of Amelogenin mRNA through Amelogenin Protein-enhanced mRNA Stabilization J. Biol. Chem., October 27, 2006; 281(43): 32439 - 32444. [Abstract] [Full Text] [PDF]

This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Ikawa, T. Articles by Oida, S. Search for Related Content PubMed PubMed Citation Articles by Ikawa, T. Articles by Oida, S.